Amyloids fall into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. Our first studies were with yeast prions, focusing on Ure2p, a negative regulator of nitrogen catabolism. Its N-terminal domain (NTD) is responsible for prionogenesis, while its C-terminal domain (CTD) performs the regulatory function of the wild-type protein. The NTD is unfolded in the wild-type protein and amyloid in the prion fibril. The CTD remains folded in the fibril but is inactivated by a steric mechanism. NTDs form the fibril backbone that is surrounded by CTDs. In 2005, we published the parallel superpleated beta-structure model for amyloid fibril backbones. The model envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Structures of this kind are good candidates for some other amyloid fibrils, including amylin. Our ongoing work is aimed at testing and refining this model and investigating fibril polymorphism. In FY17 we continued to focus on amyloid fibrils of alfa-synuclein (aS). Parkinson disease (PD) is a chronic and progressive neurodegenerative disease characterized by the intra-cerebral presence of Lewy bodies- intracerebral deposits whose principal components are amyloid fibrils of aS. Normally, the 140 aa-long protein has a membrane remodeling function that we have also researched, as reported in project AR027015-19. aS is alfa-helical when associated with lipid and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. We expressed recombinant aS in E. coli, purified the protein and assembled it into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at Brookhaven National Laboratory. Our fibrils are markedly polymorphic, as in previous reports by others. Our analysis focused on a twisting fibril with an axial repeat length of 77 nm between crossovers and an average diameter of 8.6 nm. Their reconstructed cross-section resolved this fibril into two asymmetrically associated protofibrils. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise, i.e. one subunit per axial step per protofibril, consistent with a superpleated structure. The STEM images of unstained freeze-dried specimens showed two thread-like densities running along each fibril that we interpret as metal ions. These observations support the idea that metal binding promotes fibrillation and hence Lewy Body formation in PD. A paper reporting this analysis was published towards the end of the previous reporting period (A. D. Dearborn et al., J. Biol. Chem. 291:2310-8, 2016). Our continuing efforts have aimed at extending the resolution of this analysis. In particular, we have sought to identify the metal ion(s) that form the high density threads mentioned above. A priori, copper was a plausible candidate as it has been implicated in other studies in the onset or progression of PD, and, in vitro, copper ions had been shown to bind with aS with high affinity and to accelerate the production of aS amyloid fibrils. We found that our dense threads represented the binding of metal ions scavenged from the water used to prepare them, as newly assembled preparations of aS fibrils made using highly purified water showed no threads. On the other hand, when copper was applied in defined amounts to pre-assembled thread-free fibrils, dense threads were detected on some but not all of the fibril morphotypes present. The presence of copper and the absence of other metals was confirmed by ICP-MS. The observed threading implies that the copper binding sites are axially stacked. A paper reporting this work is in preparation.